Transposons are ubiquitous mobile DNA elements. In bacteria their movement can cause mutations and genome rearrangements. Many transposons encode resistance to antibiotics and their mobility is primarily responsible for the wide dissemination of multiple-drug resistance among medically- important bacteria. The study of transposons has allowed them to be developed as powerful molecular tools and to serve as paradigms for transposons and retroviruses found in higher eukaryotes. The long term objective of this research is to determine the molecular mechanism of transposition of the bacterial insertion sequence IS903. This will be achieved by developing a structure-function analysis of the IS- encoded transposase protein, which will allow the role of transposase during transposition to be characterized. In particular the roles of specific amino acid residues will be determined. These goals will be achieved by a combined approach using a genetic screen to isolate mutants of the transposase protein, biochemical assays to determine the functional defect in these mutant proteins and a physical approach to determine functional domains of the native protein. The specific aims of this proposal are to: (1) isolate derivatives of transposase with altered properties. These will include proteins defective in transposition, proteins with increased transpositional activity, suppressor mutants that have acquired the ability to recognize mutant inverted repeats and mutants that have gained the ability to work in trans. These mutant proteins will be purified for in vitro analysis. (2) develop an in vitro transposition system to study the mechanism of IS903 transposition. (3) establish biochemical assays for the different steps in transposition. These assays will be used to characterize intermediates in transposition and to determine the functional defect of mutant transposases. (4) begin a physical analysis of the protein by mapping domains of the protein involved in DNA binding and protein-protein interactions and determining the oligomeric state of the protein in the transposition reaction. By taking this approach with IS903 we can ask some fundamental questions about transpositional biology. It will allow us to address how IS903 can transpose both conservatively and replicatively, unlike most other insertion elements. It may help us understand the apparent lack of similarity between the IS903 transposase and other IS transposases by identifying critical amino acids involved in transposition. Finally, since the IS903 transposase is strongly cis-acting, this study will also provide information about the mechanism of cis preference.